Field of the Invention
The present invention is in the field of pre-clinical therapeutics. More specifically, the present invention is directed to a method of screening synthetic and natural compounds for Janus Kinase 3 (Jak3) modulating activity.
Description of the Related Art
Janus Kinases (Jaks) are a family of non-receptor tyrosine kinase with four members; Jak1, Jak2, Jak3, and Tyk2, that transduce cytokine-mediated signals through interactions with the common γ chain of the immune receptors for IL-2, 1L-5, IL-7, IL-9, and IL-15 (1). Several studies have shown that deficiency or over-activation of Jak3 in particular, results in a multitude of disorders including, autoimmunity (2), arthritis (3) allergy (4,5), cancer (6-8), diabetes (9,10), immune deficiencies (11-13), transplant rejection (14-17) and neuromuscular diseases (18). Therefore, synthetic compounds and biomolecules that modulate Jak3 functions, without compromising normal immune functions are valuable in the treatment of these diseases.
While widely expressed in different organs in both humans and mice, Jak3 shows significant expression in the cells of the epithelial and hematologic lineages (1,19-20). In agreement with this distribution, abnormal Jak3 activation is associated with human hematologic and epithelial malignancies (21, 22).
Pharmacological inhibitors of Jak3 have shown utility in autoimmune disorders, acute lymphoblastic leukemia, Type 1 diabetes, rheumatoid arthritis, allergy and asthma. While, many of these have demonstrated promise in clinical trials, there is a need to develop sensitive, selective and reproducible tools for identifying synthetic and natural compounds that modulate Jak3 function.
The N-terminal region of Jak3 (about 550 amino acids) is known to interact not only with the cytoplasmic tail of the cytokine receptors discussed above, but also to cytoskeletal and adapter proteins. This region further contains a four-point-one ezrin/radixin/moesin (FERM) domain (23) that is implicated in cell-cell communication and cell adhesion (24), important contributors to cell motility. Thus, targeting the FERM domain is expected to inhibit kinase activity and in addition, disrupt Jak3/cytokine receptor engagement. The present invention offers a high-throughput screening method to rapidly identify compounds in tandem, for their ability to interact with the FERM domain and/or the kinase domain, with desired end use as therapeutics in the management of Jak3-associated diseases.
Currently available methods for identifying Jak3 regulators employ the kinase domain of Jak3 in two popular formats viz; (a) Caliper format and (b) Perkin Elmer format. Caliper's mobility shift assay (Caliper Life Science, Hopkinton, Mass.) uses a nanofluid-based technology, which involves electrophoretic separation of fluorescently labeled phosphorylated and non-phosphorylated substrates on a microchip, followed by fluorimetric quantification. Additionally, the reactions are run on a chip or in micro-plate wells with the microchip being used solely to separate the substrate from the phosphorylated product. The main limitation of this technology is use of external peptide substrate and the need to screen an efficient substrate that can fit into this technology. Screening of these substrates introduces yet another layer of complexity, in addition to being expensive on account of instrument and manpower costs.
Perkin Elmer's (Waltham, Mass.) Streptavidin-coated Flashplate radiometric assay differs from Caliper's assay in the use of a radiolabeled external substrate. This method uses one of three ways to assess phosphorylation: (1) measuring ATP depletion (easy-Lite-Kinase), (2) direct measurement of phosphate incorporation in the substrate using 33P-labeled ATP (FLASHPLATE), or (3) capture and measurement of phosphorylated substrate (ALPHASCREEN, Lance, DELFIA).
Another technology marketed by Dynamii Pharmaceticals (DynamixFit) also uses the kinase-only domain of Jak3, but rather than using the classical “lock and key” enzyme model, it utilizes an “induced fit” model, which is based on the concept that, enzymes being flexible proteins, have 3D active-site structures that are continuously reshaped by interactions with their substrates (or inhibitors).
Overall, there is a deficiency in the art for optimal screening tools, due to limitations imposed by use of kinase-only domains and external substrates, that is further complicated due to sensitivity, need for radioactive detection, absence of a user-friendly interface and poor cost-effectiveness. Thus, there is a recognized need for improved methods to screen Jak3 regulators. The present invention fulfills this longstanding need and desire in the art.